Growing up on a farm in northern Illinois, David Clayton decided that the country doctor had a more interesting job than the other people he encountered. So after high school, he became a chemistry major at Northern Illinois University, thinking he was en route to medical school. But the professor who taught him biochemistry during his first semester thought he would be happier in a lab. That professor must have had second sight because Clayton has enjoyed research for 48 years. He studies mitochondria, the organelles that make energy available to cells and regulate vital processes, such as cell death. He is now a lab leader at the Janelia Farm Research Campus.
Clayton's first research was in the biochemistry professor's lab, where he centrifuged enzyme preparations. Because the lab couldn't afford a refrigerated machine, Clayton had to use a bench centrifuge on the roof, where the winds of northern Illinois guaranteed low temperatures for both the enzyme and the fledgling chemist.
By the time Clayton finished his bachelor's degree, he had a paper in the Journal of Organic Chemistry. In 1965, he was accepted into graduate school at Caltech, where he studied biophysics as well as chemistry. "I believe now that chemistry is the best preparation for quantitative biology," he says, admitting that he thought he still might go to medical school after obtaining his Ph.D. But he happened to enter Jerome Vinograd's lab shortly after a new method for obtaining very pure preparations of DNA had been devised. Moreover, it was possible to separate long strands of DNA, such as those in eukaryotic nuclei, from circles of DNA, such as those in some viruses, and therefore to distinguish DNA from different sources.
By 1966, there were suggestions that mitochondria might contain DNA, but some people thought it was just a contaminant. Clayton therefore used the new technique to look for DNA in mitochondria, figuring that if it worked for viruses, it might also work for cellular organelles. The material he isolated behaved like DNA in laboratory tests. And when Clayton examined it under the electron microscope, he saw a collection of circles, each 5 microns in contour length (1/12th the diameter of a human hair), with some looped together. "I knew this was a breakthrough because you just don't find things that are all the same size in a purified preparation unless they belong there," he recalls.
Some people were not convinced that mitochondrial DNA (mtDNA) from cultured cells was the same as that in living organisms. So as part of his Ph.D. project, Clayton isolated cells from rabbits, guinea pigs, and mice, and also studied human bone marrow cells. In every cell's mitochondria, he found 5-micron circles of DNA. But when he isolated mtDNA from white blood cells of a woman with leukemia, he also saw circles that were twice as big as usual. Initially, Clayton thought his technique was faulty. "But the answer was that in leukemia cells, much of the DNA is double-sized," he says. "This was the first example of a change in mtDNA being associated with a pathological condition." More than 200 diseases involving mitochondria have now been identified, making basic studies of mitochondria very relevant to medicine.
In 1970, Clayton joined the Stanford University faculty, having abandoned any thoughts of medical school. He then began to determine how mtDNA copies itself as a mitochondrion splits in two and how its genes are transcribed into proteins. Among his many discoveries during his 26 years at Stanford was the identification of the genes involved in initiating mtDNA replication and the critical mtDNA sequences that are the targets of these gene products. He also showed that replication can't begin unless the DNA is primed by a short length of RNA. Moreover, he found that mouse mitochondrial genes have a novel genetic code. "Every time I thought we could leave mtDNA, something else would pop up," Clayton says.
By 1981, both Clayton's group and a competing group in England had sequenced the entire mouse and human mitochondrial genomes, which contain nearly 17,000 bases (as opposed to 3 billion in our chromosomes). Clayton's students and postdocs were the only sequencing group at Stanford at that time, and by the project's end they were deciphering more than 1,000 bases per month—a record at that time. Those years were among the most satisfying of Clayton's career. "It was a wonderful time because we had this orchestrated so that everybody had their part," Clayton says.
The group was also studying individual genes, including the gene for yeast mitochondrial RNA polymerase, an enzyme that moves along DNA synthesizing complementary strands of RNA. They expected the gene's sequence to resemble that of the corresponding bacterial enzyme because mitochondria appear to have originated from bacteria that set up shop inside ancient cells. But when their computer checked the gene's sequence against a database of RNA polymerases (which took several days back in the 1980s), they were surprised that it found no match to a bacterial polymerase. Instead, the yeast sequence matched that of an RNA polymerase belonging to a bacteriophage (a virus that infects bacteria). "The inescapable conclusion—and this has held up for most other mitochondrial RNA polymerases—is that the enzyme is not bacterial but is related to a viral enzyme," Clayton says, noting that this has been his most surprising discovery to date. "I don't think anyone was thinking along these lines."
The group's subsequent studies of mitochondrial RNA polymerase identified two other proteins that are essential parts of the enzyme complex. The first activates transcription by recognizing a regulatory region of DNA. By inactivating the corresponding nuclear gene in mice, the group proved the protein to be essential for fetal development, because the mice died before birth. This was the first example of a knockout mouse with a defect in a critical mitochondrial biogenesis protein.
Determining the normal functions of mitochondrial genes and what happens if they malfunction is essential to understanding diseases involving mitochondria. One of the more common is MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) syndrome, which affects cognitive and motor development and involves neuromuscular symptoms. MELAS patients were known to have an error in one of the mtDNA sequences involved in protein synthesis. But in 1991, Clayton's group showed that this mutation is embedded in another gene that helps manufacture part of the mitochondrial protein-synthesizing machinery. They then demonstrated that one part of this machinery may be overproduced when the MELAS mutation is present. Studies in other labs are determining how this glitch might cause MELAS symptoms.
Since 1991, Clayton has continued to dissect out the details of mitochondrial replication and transcription and to explore the structures of the leading players. At present, his group is developing fluorescent tags for mitochondrial proteins so they can explore mitochondrial structure in more detail, using a high-resolution imaging system that recently became available. One of the questions they will address is how mitochondrial genomes are distributed around the cell and whether this distribution differs with cell type. Another goal is to explore the structure of the nucleoid—a loose association of mtDNA, RNA, and certain proteins. Mitochondria trade information with other parts of the cell, and nucleoids are likely participants.